RESUMO
This study aimed to establish a method for the detection and identification of H7N9 avian influenza viruses based on the NA gene by pyrosequencing. According to the published NA gene sequences of the avian influenza A (H7N9) virus, a 15-nt deletion was found in the NA gene of H7N9 avian influenza viruses. The 15-nt deletion of the NA gene was targeted as the molecular marker for the rapid detection and identification of H7N9 avian influenza viruses by pyrosequencing. Three H7N9 avian influenza virus isolates underwent pyrosequencing using the same assay, and were proven to have the same 15-nt deletion. Pyrosequencing technology based on the NA gene molecular marker can be used to identify H7N9 avian influenza viruses.
Assuntos
Animais , Sequência de Bases , Aves , Galinhas , Sequenciamento de Nucleotídeos em Larga Escala , Métodos , Subtipo H7N9 do Vírus da Influenza A , Classificação , Influenza Aviária , Virologia , Dados de Sequência Molecular , Neuraminidase , Genética , Filogenia , Doenças das Aves Domésticas , Virologia , Proteínas Virais , GenéticaRESUMO
Schmallenberg virus (SBV), a novel orthobunyavirus, was first isolated in 2011. SBV preferentially infects the central nervous system of cattle and sheep and causes fever, diarrhea, a drop in milk yields, congenital malformations and stillbirths. Until June 2014, more than 200 scientific publications regarding SBV have been published. Although more than 20 articles on SVB were published in China, most of these articles provided only a brief introduction of the disease without fully discussing the associated disease characteristics. As a new disease, it has been made a focus of the National Research Center for Exotic Animal Diseases at the China Animal Health and Epidemiology Center. In this review, in order to provide a reference for research into SBV in China, we have reviewed the state of current research progress on the etiology, diagnosis and epidemiology of SBV, and vaccine development.
Assuntos
Animais , Bovinos , Infecções por Bunyaviridae , Diagnóstico , Epidemiologia , Virologia , China , Epidemiologia , Cabras , Especificidade de Hospedeiro , Orthobunyavirus , Classificação , Genética , Fisiologia , OvinosRESUMO
Based on the genomic sequence of NDV08-004 strain (GenBank accession number FJ794269), seven pairs of primers were designed to amplify the genomic fragments by RT-PCR and cloned into pGEM-Teasy vector. The fragments (named A to G) were sub-cloned into transcription vector pOLTV5 according to the universal RE site and the plasmid named NDV08-004-pO which contained the full length cDNA of NDV08-004 strain was constructed. Three helper plasmids (pCI-NP, pCI-P and pCI-L) together with NDV08-004-pO were co-transfected into BSR T7/5 cells, and the transfection supernatant was inoculated into SPF embryonated eggs to rescue the virus. The virus was rescued successfully and identified by HA and RT-PCR and sequencing. The rescue system constructed in this study provided a good foundation for the further related research.
Assuntos
Animais , Embrião de Galinha , Sequência de Bases , Vetores Genéticos , Genética , Dados de Sequência Molecular , Doença de Newcastle , Virologia , Vírus da Doença de Newcastle , Genética , Plasmídeos , Genética Reversa , MétodosRESUMO
Mutation in any of five key amino acid residues (at positions 26, 27, 30, 31 and 34) within the M2 protein of influenza A viruses leads to resistance against the amantodine class of anti-influenza drugs. In this study, a pyrosequencing method was described to rapidly detect established five molecular markers of resistance to M2 blockers, amantadine. The residues L26, V27, A30, S31 and G34 in the M2 protein were targeted for pyrosequencing, and 94 avian influenza viruses were used to perform the amantadine resistance analysis. Our results showed that most of avian influenza viruses were amantadine resistant, Mutations V27I and S31N were founded in these isolates.
Assuntos
Animais , Amantadina , Usos Terapêuticos , Antivirais , Usos Terapêuticos , Galinhas , Farmacorresistência Viral , Genética , Vírus da Influenza A , Genética , Influenza Aviária , Tratamento Farmacológico , Virologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Thirteen isolates of Class I Newcastle disease virus obtained from healthy poultry in China during 2008 were characterized genotypically in this study. All the isolates were proved to be lentogenic strains based on the deduced amino acid sequence of the Fusion protein gene. Molecular epidemiological analysis showed that 13 isolates could be subdivided into 2 distinct genotypes, 11 isolates belonged to genotype 2, and other 2 isolates belonged to genotype 3. Results indicated two genotypes of Class I Newcastle disease virus might widely exist in domestic poultry in China.
Assuntos
Animais , Humanos , Aves , China , Epidemiologia , Genótipo , Epidemiologia Molecular , Métodos , Doença de Newcastle , Epidemiologia , Virologia , Vírus da Doença de Newcastle , Classificação , Genética , Virulência , Filogenia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Virais de Fusão , GenéticaRESUMO
One highly pathogenic strain of avian influenza virus (AIV) was isolated from goose in China recently, designated as F-3. In order to study the viral entry mechanisms, the hemagglutinin (HA) gene of H5N1 subtype AIV isolate was amplified by RT-PCR, and then cloned into pGEM-T vector and sequenced. The sequencing result has logging in GenBank, the accession number was AY639405. The HA gene of F-3 had a complete open reading frame (ORE) and composed of 1707 nucleotides, coding for 568 amino acids. The deduced amino acid sequence at the cleavage site of the HA protein was RKKR GLF, matched to the characteristic of virulent avian influenza strain. The HA gene were subcloned into pcDNA3, so the plasmid pcDNA-HA can express the HA glycoprotein. Co-transfected pcDNA-HA, pHIT60 (include Murine Leukemia Virus structural genes, namely gag and pol) and pHIT111 (retroviral vector genome,containing LacZ as a reporter) into 293T cells. The retroviral supernatant were harvested 48 hours post-transfection, filtered through 0.45 micromol/L filter. The supernatant were used to analysis the characteristic of the pseudotyping virions by Western blotting and infection test. Western blotting revealed the HA glycoproteins can be expressed on the virions, indicated the glycoproteins were incorporated onto the retroviral virions. Infection test were performed on 293T, NIH3T3 and COS-7, all the three kinds of cells infected were lacZ positive, indicating viral entry, and revealed the pseudotype virions of MuLV-HA were infectious. So the pseudotype system of MuLV particles with AIV Hemagglutinin proteins were setted up and it can be used to study the entry of avian influenza virus isolated from goose in China.
Assuntos
Animais , Clonagem Molecular , Glicoproteínas de Hemaglutininação de Vírus da Influenza , Genética , Virus da Influenza A Subtipo H5N1 , Genética , Metabolismo , Vírus da Leucemia Murina , Genética , Metabolismo , Dados de Sequência Molecular , Fases de Leitura Aberta , Genética , Proteínas Recombinantes , Genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Vírion , GenéticaRESUMO
The purpose of this study was to investigate the electrophysiological effects of resveratrol on guinea pig papillary muscles and the underlying mechanism. Action potentials were recorded by using intracellular microelectrode technique. The results obtained are as follows: (1) In normal papillary muscles, resveratrol (30, 60, and 120 micromol/L) shortened the duration of action potential (APD) in a concentration-dependent manner. (2) In partially depolarized papillary muscles, resveratrol (60 micromol/L ) not only shortened APD, but also decreased the amplitude of action potential (APA), overshoot (OS) and maximal rate of depolarization in phase 0 (Vmax). (3) Perfusion with Ca2+-free K-H solution, completely abolished the effects of resveratrol (60 micromol/L) on papillary muscles. (4) Application of potassium channel blocker tetraethylammonium chloride (TEA, 20 mmol/L) did not prevent the effect of resveratrol (60 micromol/L) on action potential. (5) Pretreatment with NG-nitro-L-arginine methyl ester (L-NAME, 1 mmol/L), a nitric oxide (NO) synthase inhibitor, failed to abolish the effect of resveratrol (60 micromol/L). All these results indicate that the electrophysiological effects of resveratrol on guinea pig papillary muscles are likely due to the reduction of calcium influx, which might not be mediated by NO.